Subzero winter temperatures pose a significant challenge to the survival of organisms in temperate and polar regions. In response, many poikilothermic organisms living in these areas, including fishes, amphibians, reptiles, arthropods, plants, fungi and bacteria, have evolved physiological adaptations to survive subzero temperatures. In a given organism, multiple physiological adaptations permit cold hardiness, including, but not limited to, a subset of the following: accumulation of small molecular mass antifreezes and cryoprotectants, such as sugars and polyhydric alcohols; production of large molecular mass “antifreezes” (e.g., antifreeze proteins (AFPs)); dehydration; production of protein ice nucleators; removal of ice nucleators; and membrane adaptation.
The suite of adaptations that promotes cold hardiness in a given species leads to one of the following overwintering strategies: freeze tolerance or freeze avoidance. Freeze-tolerant organisms survive the formation of extracellular ice, but typically do not survive intracellular freezing. In contrast, freeze-avoiding organisms must avoid freezing or death will result. However, exceptions to this freeze tolerance/freeze avoidance dichotomy are known, including some insect species that can switch overwintering strategies from year-to-year, within a single winter, or even employ both strategies simultaneously in different body compartments. Paradoxically, these alternative overwintering strategies share many of the same physiological adaptations. For example, both freeze-tolerant and freeze-avoiding organisms commonly accumulate polyhydric alcohols and/or large molecular mass antifreezes during cold acclimatization.
The functions of large molecular mass antifreezes, namely antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), are best understood in freeze-avoiding organisms, where these molecules act as “antifreezes” that prevent inoculative freezing and stabilize the supercooled state through the inactivation of ice nucleators. These functions of antifreeze (glyco)proteins (AF(G)Ps) are thought to arise from their ability to interact with the surface of ice crystals and small ice-like clusters of water molecules (embryo crystals) organized by ice nucleators, and thereby inhibit their growth. This adsorption-inhibition mechanism allows AF(G)Ps to depress the freezing point of an ice crystal without significantly affecting its melting point, thus, producing thermal hysteresis (TH), a difference between the melting and freezing points of the ice crystal that is diagnostic for the presence of AF(G)Ps.
Even though TH has been described in multiple freeze-tolerant species, including representative insects, plants, nematodes and fungi, the functions and, in many cases, the chemical structures of the responsible large-molecular-mass antifreezes remain unknown. The modest levels of TH measured, typically 0.2° C.-0.5° C. or less (Duman et al., J. Insect. Physiol. 2004, 50: 259-266; Griffiths and Yaish, Trends Plant Sci. 2004, 9:399-405), do not appear to prevent the formation of ice. In fact, many freeze-tolerant organisms typically exhibit adaptations that promote freezing at high subzero temperatures, such as extracellular ice-nucleating proteins (Zachariassen and Hammel, Nature 1976, 262:285-287). Furthermore, other freeze-tolerant species of arthropods and plants may not exhibit measureable TH in spite of producing large-molecular-mass antifreezes, and instead have only pronounced hexagonal crystal growth and/or recrystallization inhibition, indicating the presence of a low activity and/or low concentration of large-molecular-mass antifreeze.
Even at very low concentrations AF(G)Ps inhibit the recrystallization of ice, thus potentially preventing damage associated with the growth of extracellular ice crystals after the initial freeze. In addition to their “antifreeze” properties, AF(G)Ps possess other functions that rely on their ability to interact with cell membranes. For instance, fish AFGPs and type I AFPs appear to protect cell membranes against the destabilizing effects of low temperatures. AF(G)Ps may also be capable of preventing propagation of ice from the extracellular fluid into the cytosol.
Accordingly, there is a need for the identification and isolation of additional biomolecules that provide thermal hysteresis (TH) properties, and for the structural determination of additional thermal hysteresis factors (THFs). There is also a need for biomolecules that can be applied to human and agricultural uses, such as in compositions that provide cold resistance and freeze resistance properties.